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Adenoviral vector pAd-E2-pIL-2 and pAd-pIL-2 Construction and Immune Effect Study

Author: HeLei
Tutor: ZhangYanMing
School: Northwest University of Science and Technology
Course: Preventive Veterinary Medicine
Keywords: Classical swine fever virus E2 gene pIL-2 gene Adenovirus
CLC: S852.5
Type: Master's thesis
Year: 2009
Downloads: 92
Quote: 0
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Abstract


Classical swine fever (Classical swine fever, CSF) is a classical swine fever virus (Classical swine fever virus, CSFV) caused by an acute, febrile, and highly lethal infectious diseases, to the pig industry has caused tremendous economic losses. In our country, classical swine fever is still very serious harm, swine fever and classical swine fever epidemic showed atypical swine fever, persistent infection and latent infection coexistence characteristics that make swine fever prevention and control more difficult and complicated. Prevention and control of swine fever vaccination is still the primary means of research can identify the natural infection and vaccination of swine fever genetically engineered vaccine is currently the focus of classical swine fever vaccine development and hotspots. Classical swine fever virus E2 protein is to stimulate the body to produce protective pigs in the main antigens and antibodies, but also in vaccine research in genetic engineering the most studied classical swine fever virus structural proteins. Expression of this study was to construct porcine interleukin 2 (Porcine interleukin 2, pIL-2) expression adenoviral vectors and expression of CSFV E2 gene and pIL-2 adenovirus vector, the purpose is to find a good immunogenic , animals can induce protection against the E2 protein of adenovirus antibodies vaccines and immune enhancing adjuvant effect of cytokines, the classical swine fever live adenovirus vector vaccine development basis. (A) the expression pIL-2 adenovirus vector and identification. First, using RT-PCR method after concanavalin respectively, from whole blood stimulated pig lymphocytes pIL-2 gene was amplified, after sequencing, and GenBank, the pIL-2 homology is 100% submission GenBank, get serial numbers FJ543108, then pIL-2 was cloned into the adenovirus shuttle vector pAdTrack-CMV to construct a recombinant adenovirus shuttle plasmid pAdTrack-pIL-2, with Pme Ⅰ endonuclease pAdTrack-pIL-2 plasmid enzyme tangent linearization, recovered transformation vector containing the adenovirus backbone pAdEasier-1 in BJ5183 competent cells, shuttle vectors and homologous recombination vector backbone, was identified recombinant adenovirus backbone vector pAdEasy-pIL-2. Digested with Pac Ⅰ and recovered pAdEasy-pIL-2, using liposome-mediated transfection of HEK293 cells were obtained recombinant adenovirus containing the target gene pAdEasy-pIL-2. PCR and RT-PCR analysis confirmed that the recombinant adenovirus can stabilize carry pIL-2 gene. After continuous passage amplification to detect the titer of recombinant adenovirus can reach 108. 25 pfu / mL. (2) co-expression of E2 gene of classical swine fever and pIL-2 adenovirus vector and identification. First, were amplified by PCR pIL-2 gene and the E2 gene, through an intermediate shared primers E2 gene and pIL-2 connection, and then the E2-pIL-2 was cloned into the adenovirus shuttle vector pAdTrack-CMV in the recombinant adenovirus shuttle plasmid pAdTrack-E2-pIL-2, with the endonuclease Pme Ⅰ pAdTrack-E2-pIL-2 was linearized plasmid was recovered after transformation vector containing the adenoviral backbone pAdEasier-1 E. coli BJ5183 competent cells shuttle vectors and homologous recombination vector backbone, was identified recombinant adenovirus backbone vector pAdEasy-E2-pIL-2. Digested with Pac Ⅰ and recovered pAdEasy-E2-pIL-2, through liposome-mediated transfection of HEK293 cells were obtained recombinant adenovirus containing the target gene pAdEasy-E2-pIL-2. After PCR, RT-PCR and Western blot analysis confirmed stable recombinant adenovirus carrying E2-pIL-2 gene. After continuous passage amplification to detect the titer of recombinant adenovirus can reach 108.12 pfu / mL. (3) the protection of recombinant rabbit test. With the constructed recombinant adenovirus pAd-pIL-2, expression of E2 gene of CSFV Shimen strain and pIL-2 adenovirus pAd-E2-pIL-2 rabbits immunized subcutaneously twice (interval 14 d), immunization dose are 2 × 107 pfu. Let both non-recombinant attenuated vaccination group and positive control group of rabbits. Free 14d after two pigs lapinized attenuated vaccine attacks, body temperature and body through the rabbit antibodies alone immune pAd-E2-pIL-2 and with pAd-pIL-2 common Rabbit fever immunization and have some ability to protect for classical swine fever live vector vaccine research and development foundation.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Serological
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